施肥和淹水管理对水稻土氨氧化微生物数量的影响
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摘要
全程氨氧化细菌(Completeammoniaoxidizers,Comammox)的发现被认为是氮循环研究的重要进展,但复杂土壤中Comammox与氨氧化细菌(Ammonia-oxidizing bacteria, AOB)和氨氧化古菌(Ammonia-oxidizingarchaea,AOA)共存的环境驱动机制尚不清楚。针对紫色水稻土长期定位试验的植稻淹水(夏季植稻施肥并且全年淹水)管理、休耕冬干(不植稻、不施肥,仅在夏季植稻期间淹水,冬季排水落干)管理,研究了施肥和水分管理对水稻土硝化潜势及氨氧化微生物类群丰度的影响。结果表明,植稻淹水土壤的硝化潜势显著高于休耕冬干,分别为25.0 mg·kg~(-1)·d~(-1)、2.11mg·kg~(-1)·d~(-1),相差可达12倍之多。实时荧光定量PCR分析表明,两种管理方式下水稻土中均能检测到Comammox、AOA和AOB,并且其数量均为Comammox>AOA>AOB。植稻淹水中Comammox丰度分别为AOA的8.5倍、AOB的77.3倍,而休耕冬干中Comammox丰度分别为AOA的4.1倍、AOB的490.3倍。相比于休耕冬干管理,植稻淹水刺激了Comammox分支A(Clade A)、AOA和AOB的生长,三者增长倍数分别为9、3、42,但Comammox分支B(Clade B)的降幅高达两倍之多。这些结果表明,与休耕冬干管理相比,28年长期植稻淹水,可能导致水稻土氨氧化微生物类群长期处于低O_2胁迫,并选择性促进了Comammox Clade A和AOA的生长,高强度施肥则显著促进了AOB生长,而Comammox Clade A和AOA则具有更广的铵态氮底物适应范围。未来应通过稳定性同位素示踪DNA技术,明确水稻土中数量上占优势的Comammox的功能意义及其与AOA和AOB的相对重要性。
The discovery of Comammox(complete ammonia oxidizers)is considered to be an important progress in the study on nitrogen recycling, but further efforts need to be done to elucidate environmental driving mechanisms of the coexistence of Comammox with AOA(ammonia-oxidizing archaea)and AOB(ammonia-oxidizing bacteria)in the complex soil environment. Soil samples were collected from a long-term stationary experiment on purple paddy soils, which had two treatments, i.e. PF(plant rice in summer and keep the field flooded all the year) and FD(fallow all the year and keep the field dried up in winter), for analysis of effects of fertilization and water management on nitrification potential and abundance of ammonia-oxidizing microorganisms in paddy soils. The analysis shows that PF was higher than FD in nitrification potential, which reached 25.0 mg· kg~(-1)·d~(-1) and 2.11 mg·kg~(-1)·d~(-1), respectively.Obviously, the former was 12 times as high as the latter. Real-time quantitative PCR(Q-PCR) shows that Comammox, AOA and AOB were detected in the paddy soils of PF and FD, and displayed an order of Comammox> AOA>AOB in abundance. The abundance of Comammox was 8.5 times that of AOA and 77.3 times that of AOB in the PF paddy soil, and 4.1 times that of AOA and 490.3 times that of AOB in the FD paddy soil. Compared with FD, PF stimulated growth of the Comammox Clade A, AOA and AOB in the soil, making the three 9, 3 and 42 times higher, respectively, in population. But the Comammox Clade B dropped 2 times as fast. All the findings in the 28 year-long experiment indicate that compared with FD, PF keeps ammonia-oxidizing microorganisms in the paddy soils under the long-term stress of O2 deficiency and selectively promotes growth of the Comammox Clade A and AOA in the soils, while fertilization at a high rate significantly promotes growth of the AOB; and Comammox Clade A and AOA can adapt to a wide range of ammonium nitrogen substrates. In future, the technique of DNA-SIP(stable isotope probe) may be used to identify functional significance of Comammox, which is dominant in population in paddy soils and its relative importance to AOA and AOB.
The discovery of Comammox(complete ammonia oxidizers)is considered to be an important progress in the study on nitrogen recycling, but further efforts need to be done to elucidate environmental driving mechanisms of the coexistence of Comammox with AOA(ammonia-oxidizing archaea)and AOB(ammonia-oxidizing bacteria)in the complex soil environment. Soil samples were collected from a long-term stationary experiment on purple paddy soils, which had two treatments, i.e. PF(plant rice in summer and keep the field flooded all the year) and FD(fallow all the year and keep the field dried up in winter), for analysis of effects of fertilization and water management on nitrification potential and abundance of ammonia-oxidizing microorganisms in paddy soils. The analysis shows that PF was higher than FD in nitrification potential, which reached 25.0 mg· kg~(-1)·d~(-1) and 2.11 mg·kg~(-1)·d~(-1), respectively.Obviously, the former was 12 times as high as the latter. Real-time quantitative PCR(Q-PCR) shows that Comammox, AOA and AOB were detected in the paddy soils of PF and FD, and displayed an order of Comammox> AOA>AOB in abundance. The abundance of Comammox was 8.5 times that of AOA and 77.3 times that of AOB in the PF paddy soil, and 4.1 times that of AOA and 490.3 times that of AOB in the FD paddy soil. Compared with FD, PF stimulated growth of the Comammox Clade A, AOA and AOB in the soil, making the three 9, 3 and 42 times higher, respectively, in population. But the Comammox Clade B dropped 2 times as fast. All the findings in the 28 year-long experiment indicate that compared with FD, PF keeps ammonia-oxidizing microorganisms in the paddy soils under the long-term stress of O2 deficiency and selectively promotes growth of the Comammox Clade A and AOA in the soils, while fertilization at a high rate significantly promotes growth of the AOB; and Comammox Clade A and AOA can adapt to a wide range of ammonium nitrogen substrates. In future, the technique of DNA-SIP(stable isotope probe) may be used to identify functional significance of Comammox, which is dominant in population in paddy soils and its relative importance to AOA and AOB.
引文
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[6]Prosser J I,Nicol G W.Archaeal and bacterial ammonia-oxidisers in soil:the quest for niche specialisation and differentiation.Trends in Microbiology,2012,20(11):523-531
[7]Martens-Habbena W,Berube P M,Urakawa H,et al Ammonia oxidation kinetics determine nicheseparation of nitrifying Archaea and Bacteria.Nature,2009,461(7266):976-979
[8] Lu L,Han W,Zhang J,et al.Nitrification of archaeal ammonia oxidizers in acid soils is supported by hydrolysis of urea.ISME Journal,2012,6(10):1978-1984
[9]Jia Z J,Conrad R.Bacteria rather than Archaea dominate microbial ammonia oxidation in an agricultural soil.Environmental Microbiology,2009,11(7):1658-1671
[10]Xia W,Zhang C,Zeng X,et al.Autotrophic growth of nitrifying community in an agricultural soil.ISME Journal,2011,5(7):1226-1236
[11]Jiang X J,Hou X Y,Zhou X,et al.pH regulates key players of nitrification in paddy soils.Soil Biology&Biochemistry,2015,81:9—16
[12]Wang B,Zhao J,Guo Z,et al.Differential contributions of ammonia oxidizers and nitrite oxidizers to nitrification in four paddy soils.ISME Journal,2015,9(5):1062-1075
[13]Kits K D,Sedlacek C J,Lebedeva E V,et al.Kinetic analysis of a complete nitrifier reveals an oligotrophic lifestyle.Nature,2017,549(7671):269-272
[14] Chu H,Fujii T,Morimoto S,et al.Community structure of ammonia-oxidizing bacteria under long-term application of mineral fertilizer and organic manure in a sandy loam soil.Applied and Environmental Microbiology,2007,73(2):485-491
[15]Rotthauwe J H,Witzel K P,Liesack W.The ammonia monooxygenase structural gene amoA as a functional marker:Molecular fine-scale analysis of natural ammonia-oxidizing populations.Applied and Environmental Microbiology,1997,63(12):4704—4712
[16]Beman J M,Francis C A.Diversity of ammoniaoxidizing archaea and bacteria in the sediments of a hypernutrified subtropical estuary:Bahia del Tobari,Mexico.Applied and Environmental Microbiology,2006,72(12):7767-7777
[17]Pjevac P,Schauberger C,Poghosyan L,et al.AmoA-targeted polymerase chain reaction primers for the specific detection and quantification of comammox Nitrospira in the environment.Frontiers in Microbiology,2017,DOI:10.3389/fmicb.2017.01508
[18]Thion C,Pjevac P,Meng A,et al.Determining the influence of soil pH on the abundance,diversity andactivity of nitrifiers:Ammonia oxidisers,nitrite oxidisers and comammox.Poster,2017,DOI:10.13140/RG.2.2.24628.55680
[19]Szukics U,Abell G C J,Hodl V,et al.Nitrifiers and denitrifiers respond rapidly to changed moisture and increasing temperature in a pristine forest soil.FEMS Microbiology Ecology,2010,72(3):395—406
[20] Szukics U,Hackl E,Zechmeister-Boltenstern S,et al.Rapid and dissimilar response of ammonia oxidizing archaea and bacteria to nitrogen and water amendment in two temperate forest soils.Microbiological Research,2012,167(2):103-109
[21]Verhamme D T,Prosser J I,Nicol G W.Ammonia concentration determines differential growth of ammonia-oxidising archaea and bacteria in soil microcosms.ISME Journal,2011,5(6):1067—1071
[22]Wertz S,Leigh A K K,Grayston S J.Effects of long-term fertilization of forest soils on potential nitrification and on the abundance and community structure of ammonia oxidizers and nitrite oxidizers.FEMS Microbiology Ecology,2012,79(1):142-154
[23]钟文辉,蔡祖聪,尹力初,等.种植水稻和长期施用无机肥对红壤氨氧化细菌多样性和硝化作用的影响.土壤学报,2008,45(1):105—111Zhong W H,Cai Z C,Yin L C,et al.Effects of rice cultivation and long term application of inorganic fertilizers on ammonium oxidizers diversity and nitrification of red soils(In Chinese).Acta Pedologica Sinica,2008,45(1):105—111
[24]Hansel C M,Fendorf S,Jardine P M,et al.Changes in bacterial and archaeal community structure and functional diversity along a geochemically variable soil profile.Applied and Environmental Microbiology,2008,74(5):1620-1633
[25]蔡祖聪,赵维.土地利用方式对湿润亚热带土壤硝化作用的影响.土壤学报,2009,46(5):795—801Cai Z C,Zhao W.Effects of land use types on nitrification in humid subtropical soils of China(In Chinese).Acta Pedologica Sinica,2009,46(5):795-801
[2]Daims H,Lebedeva E V,Pjevac P,et al.Complete nitrification by Nitrospira bacteria.Nature,2015,528(7583):504-509
[3]van Kessel M A H J,Speth D R,Albertsen M,et al.Complete nitrification by a single microorganism.Nature,2015,528(7583):555-559
[4]Pinto A J,Marcus D N,Ijaz U Z,et al.Metagenomic evidence for the presence of Comammox Nitrospiralike bacteria in a drinking water system.MSphere,2015,DOI:10.1128/mSphere.00054-15
[5]Palomo A,Jane F S,Gulay A,et al.Metagenomic analysis of rapid gravity sand filter microbial communities suggests novel physiology of Nitrospira spp.ISME Journal,2016,10(11):2569-2581
[6]Prosser J I,Nicol G W.Archaeal and bacterial ammonia-oxidisers in soil:the quest for niche specialisation and differentiation.Trends in Microbiology,2012,20(11):523-531
[7]Martens-Habbena W,Berube P M,Urakawa H,et al Ammonia oxidation kinetics determine nicheseparation of nitrifying Archaea and Bacteria.Nature,2009,461(7266):976-979
[8] Lu L,Han W,Zhang J,et al.Nitrification of archaeal ammonia oxidizers in acid soils is supported by hydrolysis of urea.ISME Journal,2012,6(10):1978-1984
[9]Jia Z J,Conrad R.Bacteria rather than Archaea dominate microbial ammonia oxidation in an agricultural soil.Environmental Microbiology,2009,11(7):1658-1671
[10]Xia W,Zhang C,Zeng X,et al.Autotrophic growth of nitrifying community in an agricultural soil.ISME Journal,2011,5(7):1226-1236
[11]Jiang X J,Hou X Y,Zhou X,et al.pH regulates key players of nitrification in paddy soils.Soil Biology&Biochemistry,2015,81:9—16
[12]Wang B,Zhao J,Guo Z,et al.Differential contributions of ammonia oxidizers and nitrite oxidizers to nitrification in four paddy soils.ISME Journal,2015,9(5):1062-1075
[13]Kits K D,Sedlacek C J,Lebedeva E V,et al.Kinetic analysis of a complete nitrifier reveals an oligotrophic lifestyle.Nature,2017,549(7671):269-272
[14] Chu H,Fujii T,Morimoto S,et al.Community structure of ammonia-oxidizing bacteria under long-term application of mineral fertilizer and organic manure in a sandy loam soil.Applied and Environmental Microbiology,2007,73(2):485-491
[15]Rotthauwe J H,Witzel K P,Liesack W.The ammonia monooxygenase structural gene amoA as a functional marker:Molecular fine-scale analysis of natural ammonia-oxidizing populations.Applied and Environmental Microbiology,1997,63(12):4704—4712
[16]Beman J M,Francis C A.Diversity of ammoniaoxidizing archaea and bacteria in the sediments of a hypernutrified subtropical estuary:Bahia del Tobari,Mexico.Applied and Environmental Microbiology,2006,72(12):7767-7777
[17]Pjevac P,Schauberger C,Poghosyan L,et al.AmoA-targeted polymerase chain reaction primers for the specific detection and quantification of comammox Nitrospira in the environment.Frontiers in Microbiology,2017,DOI:10.3389/fmicb.2017.01508
[18]Thion C,Pjevac P,Meng A,et al.Determining the influence of soil pH on the abundance,diversity andactivity of nitrifiers:Ammonia oxidisers,nitrite oxidisers and comammox.Poster,2017,DOI:10.13140/RG.2.2.24628.55680
[19]Szukics U,Abell G C J,Hodl V,et al.Nitrifiers and denitrifiers respond rapidly to changed moisture and increasing temperature in a pristine forest soil.FEMS Microbiology Ecology,2010,72(3):395—406
[20] Szukics U,Hackl E,Zechmeister-Boltenstern S,et al.Rapid and dissimilar response of ammonia oxidizing archaea and bacteria to nitrogen and water amendment in two temperate forest soils.Microbiological Research,2012,167(2):103-109
[21]Verhamme D T,Prosser J I,Nicol G W.Ammonia concentration determines differential growth of ammonia-oxidising archaea and bacteria in soil microcosms.ISME Journal,2011,5(6):1067—1071
[22]Wertz S,Leigh A K K,Grayston S J.Effects of long-term fertilization of forest soils on potential nitrification and on the abundance and community structure of ammonia oxidizers and nitrite oxidizers.FEMS Microbiology Ecology,2012,79(1):142-154
[23]钟文辉,蔡祖聪,尹力初,等.种植水稻和长期施用无机肥对红壤氨氧化细菌多样性和硝化作用的影响.土壤学报,2008,45(1):105—111Zhong W H,Cai Z C,Yin L C,et al.Effects of rice cultivation and long term application of inorganic fertilizers on ammonium oxidizers diversity and nitrification of red soils(In Chinese).Acta Pedologica Sinica,2008,45(1):105—111
[24]Hansel C M,Fendorf S,Jardine P M,et al.Changes in bacterial and archaeal community structure and functional diversity along a geochemically variable soil profile.Applied and Environmental Microbiology,2008,74(5):1620-1633
[25]蔡祖聪,赵维.土地利用方式对湿润亚热带土壤硝化作用的影响.土壤学报,2009,46(5):795—801Cai Z C,Zhao W.Effects of land use types on nitrification in humid subtropical soils of China(In Chinese).Acta Pedologica Sinica,2009,46(5):795-801